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Investigative Ophthalmology July 1976 546 Reports 7. 8. 9. 10. of gluconeogenesis in the perfused livers of rats, J. Biol. Chem. 242:2622, 1967. Thoft, R. A., and Friend, J.: Biochemical aspects of contact lens wear, Am. J. Ophthalmol. 80: 139, 1975. Riley, M. V.: Aerobic glycolysis in the ox cornea, Exp. Eye Res. 8: 201, 1969. Reddy, V. N.: Distribution of free amino acids and related compounds in rabbit cornea, Ophthalmol. Res. 1: 46, 1970. Schonheyder, F., Ehlers, N., and Hust, B.: Amino acids in aqueous humor and plasma in chronic ocular disorders, Acta Ophthalmol. 53: 627, 1975. Hydrostatic pressure effects on deswelling of de-epithelialized and de-endothelialized corneas. KAREN A. BOWMAN AND KEITH GREEN. The effect of varying hydrostatic pressure on the thinning rate of preswollen de-epithelialized or de-endothelialized corneas has been determined in the specular microscope. The appropriate membrane was removed, the cornea given access to Ringer to swell, and then fluid exchange at that surface blocked with oil. De-epithelialized corneas thin more slowly as hydrostatic pressure on the posterior surface is increased, until fluid movement ceases at 60 to 70 mm. Hg. Fluid movement can occur, therefore, against a considerable hydrostatic pressure. De-endothelialized corneas thin at a higher rate as hydrostatic pressure is increased; this effect is probably a mechanical one with increasing pressure forcing fluid out across the epithelium. Since its introduction, the specular microscope has been used extensively in the study of corneal physiology.1"3 With this system, the cornea can be excised atraumatically from the eye and mounted in appropriate chambers for determination of thickness to an accuracy of ± 2 fim. With little variation most investigators have elected to use hydrostatic pressure of about 15 mm. Hg1-3 on the endothelial surface, although lower pressures have been used, e.g., 8 mm. Hg.4 One study has reported no effect of hydrostatic pressure variation between 6 and 37 mm. Hg on the deswelling rate of the de-endothelialized cornea.5 The present work was a study of the effects of varying the applied hydrostatic pressure between 5 and 50 mm. Hg on the deswelling rate of both de-epithelialized and de-endothelialized corneas. Materials and methods. Adult albino rabbits, 2 to 3 kilograms, were killed with an overdose of sodium pentobarbital and the eyes enucleated with the lids as described previously.1 Corneal excision and mounting in the appropriate chambers also followed the standard procedure.1"3 De-epithelialized corneas. For convenience the epithelium was removed from the cornea with a Gill corneal knife prior to enucleation and mounting. The stroma, therefore, was allowed access to fluid over a time period of about 10 minutes prior to placement in the chamber. Krebsbicarbonate-Ringer's solution,1 with added adenosine (5.0 mM.) and reduced glutathione (0.3 mM.), 6 was placed on the exposed stromal surface and the stroma allowed to swell for 15 minutes after mounting; the fluid was removed and the stroma covered with silicone oil (No. 20 CSKS Dow Corning, Midland, Mich.) to prevent further fluid exchange across the exposed stromal surface. Thickness was measured by alternately focusing on the anterior stromal surface and the endothelium. The endothelial surface was perfused with modified Krebs-bicarbonate-Ringer's solution at 37° C. at a rate of 33 /tl per minute. A control series consisted of thickness measurements at 15 mm. Hg for 5 hours. The experimental series consisted of an initial 1 hour of 15 mm. Hg, for comparison with the controls, followed by one of three other subsequent pressures 5, 30, or 50 mm. Hg applied for the second hour, a different pressure from this series applied for the third hour, and the remaining unused pressure from this series applied for the fourth hour, which allowed at least four corneas to be subjected to each possible sequence of these three pressures. A final period of 15 mm. Hg followed between hours 4 and 5. Each pressure was applied for 1 hour, and the deswelling rate determined over the final 30 minutes of each hour, since initial experiments showed that the thickness adjustment became regular within 30 minutes. De-endothelialized corneas. The endothelium was removed by gentle scraping with the cornea attached to the mounting rod. The posterior surface of the cornea was given free access to Ringer's solution and allowed to swell for 20 minutes. The normal mounting procedure was then followed except that silicone oil was perfused across the exposed posterior stromal surface and the epithelial surface was perfused with modified Ringer's solution. The perfusion system delivered Ringer's at 1 ml. per minute and 32° C. to the epithelial surface and the fluid level was maintained with a vacuum overflow. The normal volume needed to fill the anterior space bordering the epithelium was 1 ml. without immersion of the objective, thus the turnover was 1 volume per minute, sufficient to prevent stagnation of the epithelial fluid bath which, by evaporation, could become more hypertonic. The objective was immersed into the perfusing solution every 30 minutes for thickness determinations. A similar experimental protocol to that outlined above for the de-epithelialized corneas was followed; that is, at least two corneas were used Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017 Reports Volume 15 Number 7 547 560 i 15 mmHg 54 5 mmHg 50 mmHg i °i-^< 520- ••30///hr*) 500H V 480- "•> \ 460H 440H 4 20 20 40 80 60 100 120 140 160 180 TIME (min.) Fig. 1. Deswelling response of de-epithelialized cornea to adjustment in applied hydrostatic pressure as a function of time. A representative of six corneas is shown to illustrate stabilization of deswelling rate within 20 minutes. Table I. Deswelling rates of de-epithelialized and de-endothelialized corneas at different pressures when compared at different time intervals" Time(hr) Pressure (mm. Hg.) De-epithelialized: 5 15 30 50 De-endo th elialized: 5 ' 15 30 50 0-1 1-2 — 28.7 36.5 14.4 8.5 18.3 ± 3.5 — +0.3 15.0 16.5 — 32.3 ± 6.8 2-3 3-4 ± 3.9 ± 1.2 ± 7.2 ± 3.1 31.3 ± 19.0 ± 20.7 ± 9.5 ± 5.7 6.9 12.1 3.5 ± 5.8 ± 2.9 ± 5.6 50.3 ± 4.7 3.8 ± 7.5 ± 12.0 ± 24.5 ± 9.9 4.8 2.5 9.7 25.0 24.8 16.0 11.3 ± ± ± ± 4-5 7.2 2.0 2.1 2.2 ± 11.4 ± 4.0 ± 3.0 27.8 ± 7.5 2.5 9.0 4.5 1.0 ± 1.9 1.4 ±2.9 0 De-epithelialized corneas: Values (/im/hr.) are the mean ± S.E. of four determinations except for 0-1 hr. and 4-5 hr. 15 mm. Hg, which represent the mean ± S.E. of 12 determinations. De-endothelialized corneas: Values are the mean ± S.E. of eight determinations except for 0-1 hr. and 4-5 hr. for 15 mm. Hg, which represents the mean ± S.E. of 24 determinations. The positive sign for 5 mm. Hg, 1-2 hr., indicates a gain in thickness. The data in the table combine both the data from the constant pressure series at 15 mm. Hg and the variable pressure data from the experimental series. at each pressure which was applied in a pattern as described above. Results. Initial experiments using six deepithelialized and 2 de-endothelialized corneas provided a time course for stabilization of deswelling after a pressure change. A typical result for a de-epithelialized cornea is shown in Fig. 1, where the effect of the pressure change per se was absorbed within 15 to 20 minutes after that change; the final 30 minutes of each hour was used, therefore, for deswelling rate determinations. A similar rapid change in response to pressure was found for de-endothelialized corneas. De-epithelialized corneas. The initial thickness of corneas after swelling was 549.3 ± 7.3 jum (16 corneas) (mean ± S.E.). The deswelling rate over the first 4 hours was found to be consistent (see Table I for 15 mm. Hg); between 4 and 5 hours, however, the rate slows from 28.1 ± 2.8 /*m per hour4 over the previous four hours to 1.0 ± 1.9 ^m per hour. At 15 mm. Hg between 4 and 5 hours, after prior exposure to the various Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017 Investigative Ophthalmology July 1976 548 Reports n i O i 15- i 5 u> 20- THINNING , s^ > 10 20 30 40 50 60 PRESSURE (mmHg) Fig. 2. Deswelling rate as a function of hydrostatic pressure applied to the posterior surface of deepithelialized corneas. X indicates the mean deswelling rate found for corneas subjected to constant 15 mm. Hg for 4 hours. Each point is the mean ± S.E. of 12 measurements. other pressures, the rate was 10.3 ± 5.6 fim per hour, due to the difference between this value and that obtained at 15 mm. Hg during the preceding 4 hours the final time period data were not utilized in the analysis. The good agreement between deswelling rates at each pressure at any time interval (Table I) allowed the data to be pooled and the result is shown in Fig. 2. The deswelling rate at 50 mm. Hg. is significantly less than that found at either 5 (0.01 > p > 0.005) or 15 mm. Hg (0.05 > p > 0.025), but not significantly different from the rate at 30 mm. Hg. De-endolhelialized corneas. This initial thickness of corneas after swelling was 505.2 ± 4.0 /mi (28 corneas). The deswelling rate at 15 mm. Hg over 4 hours was consistent (Table I) at 12.5 ± 2.1 /fin per hour. In other experiments, where another protocol was followed after either 1 or 2 hours, the deswelling rate was found to be 13.3 ± 1.6 (n = 24) pm per hour. The consistency of deswelling at each pressure at any time allowed the experimental data to be pooled. At 15 mm. Hg, after being subjected to other pressures during the preceding 4 hours, the thickness reduction was 1.4 ± 2.9 /mi per hour. Only the data obtained during the first 4 hours were used since there was such a large decrease in thinning rate after this time. The results are shown in Fig. 3, where the slope indicates that as the applied pressure is increased so the deswelling rate increases. The deswelling rate at 50 mm. Hg is significantly greater than that found at either 5, 30, or 15 mm. Hg (p > 0.05). The value at 5 mm. Hg (Fig. 3) is significantly different (p < 0.001) from that at 30 and 50 mm. Hg and the 15 and 30 mm. Hg values are significantly different from the 50 mm. Hg value (p < 0.01); the 15 and 30 mm. Hg values are not significantly different from each other. The deswelling rate and total quantity of thinning are far in excess of any significant contribution of the epithelial thickness, and must reflect stromal thickness changes. Discussion. De-epithelialized corneas. The rate of deturgescence of de-epithelialized corneas, at 15 mm. Hg, is similar to that seen by previous workers,1"1 thus the endothelia were functioning normally. The initial 15 mm. Hg deswelling rate in the series of corneas subjected to alternating pressures was the same as that determined in the corneas at constant 15 mm. Hg for 4 hours (see Table 1 and Fig. 2). The constant-pressure experiments reveal that the first 4 hours could be directly compared (Table 1, 15 mm. Hg) and that changes caused by altering the applied pressure were complete within 20 minutes (Fig. 1). The data shown in Fig. 2 indicate that at pressures greater than 15 mm. Hg there is an influence of pressure on the deswelling rate. Between 5 and 15 mm. Hg there is no significant difference in deswelling rate, thus a curve is shown with a pressure-independent component between 5 and 15 mm. Hg and a linear relationship indicated from 15 to 50 mm. Hg. Hodson'1 reported that a pressure change from 6 to 37 mm. Hg had no efFect on the deturgescence rate, although others' have reported that at about 60 to 80 mm. Hg the fluid movement across the endothelium from the stroma to aqueous humor surface is no longer able to offset the fluid leak into the cornea. The difference between these findings is perhaps related to the higher pressures used by others" and ourselves which caused a significant difference to be found in the thinning rate. By extrapolating the linear curve in Fig. 2, values for the pressure where no net flow occurs across the endothelium would be obtained of between 65 and 75 mm. Hg. The net fluid movement out of the stroma across the endothelium, therefore, can occur against a considerable hydrostratic pressure gradient. Whatever the driving force, this movement implies a considerable physiologic force exists for fluid movement across the endothelium. De-endolhelialized corneas. The deswelling rate of de-endothelialized corneas is somewhat larger than that reported in one instance^ +0.9 tun per hour, and about the same as that reported by Zadunaisky." Klyce* mentioned that de-endothe- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017 Reports 549 Volume 15 Number 7 35- "^ 30 " J. M !S . O H § 15- kj io- I M 0- b + 5- —r— 20 —r— 30 —r— 40 —I 50 PRESSURE (mmHg) Fig. 3. Deswelling rate as a function of hydrostatic pressure applied to the posterior surface of de-endothelialized corneas. X indicates the mean deswelling rate found for corneas subjected to constant is 5 mm. Hg for 4 hours. Each point is the mean ± S.E. of 12 measurements. lialized corneas deswell at about 15 nm per hour if the epithelial bathing solution is not replenished, but in our experiments the perfusion rate was 1 ml. per minute, giving a complete replacement of fluid each minute. Evaporation, with a consequent increase in osmotic pressure of the epithelial bathing solution, was not a factor, therefore, in the present experiments and must reflect activity of the epithelium. Contrary to the effect of increasing pressure on the de-epithelialized cornea, the de-endothelialized cornea deswells more rapidly as the hydrostatic pressure on the posterior surface is increased. This finding may be a consequence of tissue compression; since the cornea is held tightly between the two chambers raising the pressure on the posterior surface will effectively squeeze more fluid out of the cornea at a faster rate, especially since the relatively impermeable epithelium10 is present. It appears, therefore, that the increased water loss across the epithelium in response to elevated hydrostatic pressures is the result of a mechanical phenomenon rather than a reflection of a physiologic process. In the deepithelialized cornea, however, compression of the stroma is not a relevant explanation for the steady-state results, since the deswelling rate falls rather than increases. The endothelium is relatively leaky to water10 and as such will allow more water to pass as the hydrostatic pressure is increased and thus more fluid will be forced into the stroma against the effect of the endothelial fluid movement out of the stroma and under these conditions, the cornea will not deswell as fast. These results emphasize that, under nonstimulated conditions, both membranes remove fluid from the stroma at zero hydrostatic pressure. The fluid movement, however, occurs at a rate dependent on the stromal hydration since the rate falls markedly once a near-normal thickness is achieved. We thank Ms. Debbie Hancock for her secretarial assistance. From the Departments of Ophthalmology and Physiology, Medical College of Georgia, Augusta, Ga. Supported in part by Public Health Service Research Grant EY 01413 from the National Eye Institute. Lions Eye Bank-Augusta made the specular microscopes available. Submitted for publication Feb. 10, 1976. Reprint requests: K. Green, Ph.D., 3 D 11, R & E Building, Department of Ophthalmology, Medical College of Georgia, Augusta, Ga. 30902. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017 550 Key words: rabbit, cornea, epithelium, endothelium, hydrostatic pressure, deswelling rate, specular microscope. REFERENCES 1. Maurice, D. M.: The location of the fluid pump in the cornea, J. Physiol. 221: 43, 1972. 2. McCarey, B. E., Edelhauser, H. F., and Van Horn, D. L.: Functional and structural changes in the corneal endothelium during in vitro perfusion, INVEST. OPHTHALMOL. 12: 3. 4. 5. 6. 7. 8. 9. 10. Investigative Ophthalmology July 1976 Reports 410, 1973. Fischbarg, J.: Active and passive properties of the rabbit corneal endothelium, Exp. Eye Res. 15: 615, 1973. Coles, W. H.: Pilocarpine toxicity. Effects on the rabbit corneal endothelium, Arch. Ophthalmol. 93: 36, 1975. Hodson, S.: The regulation of corneal hydration by a salt pump requiring the presence of sodium and bicarbonate ions, J. Physiol. 236: 271, 1974 . Dikstein, S., and Maurice, D. M.: The metabolic basis to the fluid pump in the cornea, J. Physiol. 221: 29, 1972. Kaye, G. I., Sibley, R. C , and Hoefle, F. B.: Recent studies on the nature and function of the corneal endothelial barrier, Exp. Eye Res. 15: 585, 1973. Klyce, S. D.: Transport of Na, Cl and water by the rabbit corneal epithelium at resting potential, Am. J. Physiol. 228: 1446, 1975. Zadunaisky, J. A.: The control of comeal thickness by activation of epithelial transport. Paper presented at 1st International Congress of Eye Research, June 2-7, 1974, Capri, Italy. Green, K., and Green, M. A.: Permeability to water of rabbit corneal membranes, Am. J. Physiol. 217: 635, 1969. Automatic recording of corneal thickness in vitro. STEPHEN D. KLYCE AND DAVID M. MAURICE. An addition to the specular microscope is described which allows it to record the thickness of the excised cornea automatically as a function of time. The focus of the instrument is scanned mechanically through the tissue, and the position of the reflecting surfaces is detected by a photoelectric system and marked on a chart recorder. The system is able to follow thickness changes over periods of many liours and with an accuracy greater than obtainable by manual operation. This system has been helpful in the evaluation of a new medium which considerably extends the useful lifetime of the corneal endothelial fluid pump. The specular microscope1' 2 has been used extensively to measure the thickness of the excised and perfused cornea. Generally, readings are taken at intervals by a trained observer, which is an acceptable procedure when the preparation survives for only about 7 hours, as is commonly the case. However, several workers are attempting to find which are the missing factors which would extend its lifetime, and any degree of success results in inconveniently long working periods. It was evident that an automated system was called for, and this report describes an attachment to the specular microscope that has been developed for the continuous measurement of cornealthickness. Materials and methods. Under the specular microscope, readings of corneal thickness are obtained by difference on manually focusing either up or down through the cornea and recording the calibrations on the fine-focus dial when a slit of light projected through the objective is focused on its surfaces. A reflecting surface forms an image of the slit at the eyepiece, and this image shifts across the field when the microscope is moved through focus. The position of the slit on an eyepiece scale proved to be a more reproducible indicator of the position of the surface than a subjective estimate of sharpness of its image. Description of apparatus. In the automated version (Fig. 1) the focus of the microscope is scanned mechanically through the cornea while the positions of the surface reflections are sensed and recorded electronically. The rotary motion of the fine-focus dial is sensed by a ten-turn potentiometer coupled to the dial to provide a 10 mV. increment in signal for each 3.60° (equivalent to 1 /mi) of rotation. This signal is fed through a sample-and-hold module to drive the Y axis of a 10" chart recorder (Model SR-255B, Heath Co.) while the chart is driven at slow speed (0.01" per minute). Motorized vertical scan (100 /mi per minute) is accomplished by driving the fine-focus dial with a reversible 4 r.p.m. synchronous motor. The motor alternately raises and lowers the microscope body through an adjustable scan range of 50 to 950 /mi. As the microscope is scanned through the cornea, the focused images are transmitted by fiber optics to a photometric system whose output triggers the chart recorder to make a dot (by lowering the pen) corresponding to the position of the fine-focus dial each time a peak of a relatively intense reflection is sensed. As mechanical hysteresis is inherent in the microscope, a double dotted line is traced for each reflecting surface when both directions of scan are recorded. To check for accuracy and stability, the thickness of a coverglass slip was Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933299/ on 05/03/2017